G Model
ARTICLE IN PRESS
JVAC-15126; No. of Pages 4
Vaccine xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Development of a vectored vaccine against Hepatitis E virus Khaled Trabelsi a , Amine Kamen b , Héla Kallel a,∗ a Viral Vaccines Research & Development Unit, Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Institut Pasteur de Tunis, 13 Place Pasteur, BP 74 1002 Tunis, Tunisia b Animal Cell Technology, Biotechnology Research Institute, National Research Council of Canada, Montreal, Canada
a r t i c l e
i n f o
Article history: Available online xxx Keywords: Hepatitis E virus Insect cells SF9 Baculovirus Adeno-associated virus
a b s t r a c t Hepatitis E virus is a non-enveloped ssRNA virus [1] that causes human acute hepatitis through primarily fecal and oral transmission [2]. Currently, no commercial hepatitis E (HEV) vaccine is available. In the absence of an appropriate cell culture system for HEV propagation, HEV pseudocapsids (ORF2 protein) have been produced either in Escherichia coli or in insect cells and they have been shown to protect monkeys against virus challenge and to be effective in the prevention of natural HEV infection of humans. In this work, we propose to develop a novel candidate vaccine against hepatitis E infection using adenoassociated virus (AAV) as a vector expressing the gene of the truncated capsid protein of HEV (aa 112–aa 660). rAAV will be produced in Sf9 cells using the baculovirus expression vector system. For this purpose, construction of recombinant baculoviruses was performed and viral stocks of BacRep, BacCap for serotypes 2, 5 and 6 were prepared in Sf9 cells. The recombinant baculovirus coding for the truncated capsid protein of HEV (BacITRHEVORF2) was also constructed, the virus titer was equal to 5.41 × 109 PFU/mL, at the third passage. Transduction of HEK 293 EBNA cells with rAAV was carried out; the production of HEVORF2 was confirmed by Western blot. Optimization of rAAV production in Sf9 cells is currently ongoing. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction HEV is classified into the family Hepeviridae and genus Hepevirus. Based on the available nucleotide sequences, HEV isolates have been divided into five genotypes, each with a different geographical distribution. The HEV genome contains three open-reading frames (ORFs) that encode the ORF1 non-structural polyprotein with proposed biochemical functions, the ORF2 major viral capsid protein, and the ORF3 regulatory protein. Various recombinant forms of the ORF2, expressed using different heterologous systems are being explored [3]. Two candidate vaccines are in development and are being tested in clinical trials. The recombinant ORF2 proteins produced using baculovirus vectors in insect cells have been pursued vigorously as candidate vaccines. The 56-kDa ORF2 truncated protein expressed in insect cells, accumulates in the cytoplasm. This protein was found
∗ Corresponding author at: Viral Vaccines Research and Development Unit, Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Institut Pasteur de Tunis, 13 Place Pasteur, BP 74, 1002 Belvédère, Tunis, Tunisia. Tel.: +216 71 783 022; fax: +216 71 791 833. E-mail address: [email protected] (H. Kallel).
to be an efficient immunogen in monkeys when given with an alum adjuvant. Phases II–III efficacy trial conducted in young healthy adults (soldiers) in Nepal, showed an efficacy rate of 95%. The volunteers were given 20 g of the alum-adjuvanted recombinant HEV protein at 0, 1 and 6 months. However, despite such progress, the vaccine still needs several years more work before its licensing [4]. The second candidate vaccine is expressed in Escherichia coli, it is called HEV293, contains ORF2 amino acids 376–606. A large phase III trial of this vaccine was conducted in adults in China; the volunteers received three doses of HEV 239 (30 g of purified recombinant hepatitis E antigen adsorbed to 0.8 mg aluminum hydroxide suspended in 0.5 ml buffered saline) given intramuscularly at 0, 1, and 6 months. Data showed that this vaccine was safe and effective. Adverse effects attributable to the vaccine were few and mild. No vaccination-related serious adverse event was noted [5]. However both candidate vaccines need the use of an adjuvant, are delivered intramusculary and require several injections to be effective. Therefore these vaccines may have a high cost, and could not be affordable for populations living in developing countries, where HEV is endemic. In this work we suggest to develop a vectored vaccine against hepatitis E virus that will be delivered by the nasal route. For this purpose we will use the AAV as a vector to
http://dx.doi.org/10.1016/j.vaccine.2014.02.041 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Trabelsi K, et al. Development of a vectored vaccine against Hepatitis E virus. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.041
G Model JVAC-15126; No. of Pages 4
ARTICLE IN PRESS K. Trabelsi et al. / Vaccine xxx (2014) xxx–xxx
2
Fig. 1. Electrophoretic separation on 1% agarose gel. (A) T6 Rep2/Cap6 Bacmid clones #2 and #5. (B) T2 Rep2/Cap2 Bacmid clones #3 and #1. (C) BacCap5 Bacmid clones #8, #9, and #10. (Lad) DNA ladder.
deliver the antigen which is the truncated capsid protein of HEV (aa 112–aa 660). Baculovirus and insect cells platform will be used for the production of rAAV. 2. Material and methods
pH 7.4, 0.02% Tween-20, 3% skimmed milk). Membranes were incubated with the appropriate primary detection antibody for 1 h with gentle agitation, washed three times with PBST, then incubated for an additional 1 h with the appropriate peroxidase conjugated secondary antibody. Immunoreactive proteins were visualized by ECL (GE Healthcare, Uppsala, Sweden).
2.1. Cells and medium 3. Results and discussion Sf9 insect cells were used in this study. They were grown in suspension in the serum free medium SF900II (Invitrogen, The Netherlands) in polycarbonate Erlenmeyer flasks (Corning, NY, USA) at 27 ◦ C and 115–120 rpm. Cultures were seeded at 0.45–0.5 × 106 cells/mL. 2.2. Plasmid and recombinant baculovirus construction BacITRHEVORF2 was constructed using the Baculogold system from Becton Dickinson (USA). BacRep2, BacCap2, BacT2 Rep/Cap and BacT6 Rep/cap baculoviruses were constructed using the Bacto-Bac baculovirus expression system from Invitrogen (Canada). The plasmids pFBVP5, pFBDLSR Rep2, pSR657 BacT2 Rep/Cap, pSR646 Bac T6 Rep/Cap were kindly provided by Dr Kotin from the Laboratory of Biochemical Genetics (NHLB, NIH, USA). 2.3. Virus stock preparation Sf9 cells growing exponentially were infected at a density 2–2.5 × 106 cells/ml with 0.1–0.5% (v/v) of recombinant baculovirus. Virus harvests were collected after 72–96 h post-infection, culture supernatants were centrifuged at 4000 rpm, 10 min then stored at 4 ◦ C or −80 ◦ C in SF900II medium + 1% fetal bovine serum (FBS). 2.4. Estimation of virus titer Virus titer was estimated according to the MTT viability assay [6], flow cytometry [7] and the easy titer Sf9/GFP [8]. 2.5. AAV quantification Vector genome quantification was carried out by real timePCR [9]. 2.6. Western blot Samples were analyzed by SDS-PAGE under reducing conditions using 15% polyacrylamide gels and then transferred onto nitrocellulose membranes using the transfer System (Bio-Rad, USA). The membranes were then blocked overnight in PBST solution (PBS,
3.1. Construction of the vectors BacRep2, BacCap5, the dual plasmids T2 Rep2/Cap2 and T6 Rep2/Cap6 The plasmids provided by Dr Kotin were used to construct the different types of baculovirus (BacRep2, BacCap5, BacRep2/Cap2 and BacRep2/Cap6) using the Bac-to-Bac system. The expression cassette of the pFastBac vector recombines with the parent bacmid in DH10BacTM E. coli competent cells to form the expression bacmid. After extraction and purification the bacmid were analyzed by electrophoretic separation in agarose gels (Fig. 1). The bacmid were then transfected into insect cells for the production of recombinant baculovirus. 3.2. Production of recombinant baculovirus and titration Recombinant baculovirus (BacRep2, BacCap2, BacCap5, BacRep2/Cap2, BacRep2/Cap6 and BacITRHEVORF2) were amplified in Sf9 cells grown in SF900-II medium over three passages. Cells were grown at 27 ◦ C and infected at an MOI of 1 and at an initial cell density of 2 × 106 cells/mL. Once cell viability reached a level less than 60%, baculovirus were harvested by centrifugation. The harvests were also filtrated through 0.22 m, 1% fetal calf serum was added to the baculovirus stocks before being stored at −80 ◦ C. Virus titers were estimated using different methods; data obtained are shown in Table 1. Virus titer of the different baculovirus obtained at the third passage was slightly higher than 108 PFU/mL as determined by the MTT method.
Table 1 Titration of recombinant baculovirus using different methods. Baculovirus
TCID50 (PFU/mL) (using Sf-9 easy titer cell line)
FACS (PFU/mL)
MTT assay (PFU/mL)
P3 BacRep2/Cap2 P3 Bac Rep2/Cap6 P3 Bac Cap5 P3 BacCap2 P3 Bac Rep2
Nd Nd 7.64 × 107 Nd Nd
Nd Nd 1.97 × 109 Nd Nd
3.2 × 108 2.28 × 108 3.23 × 108 3.59 × 108 2.21 × 108
Nd, not determined.
Please cite this article in press as: Trabelsi K, et al. Development of a vectored vaccine against Hepatitis E virus. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.041
G Model JVAC-15126; No. of Pages 4
ARTICLE IN PRESS K. Trabelsi et al. / Vaccine xxx (2014) xxx–xxx
3
Fig. 2. (A) AAV5 capsid proteins analysis by Western blot. Detection of the Cap proteins (VP1, VP2, VP3) for clones #8, #9 and #10. (B) AAV2 replicase proteins (Rep78, Rep68, Rep52, Rep40) analysis by Western blot for clones #1, #8, T2#1, T6#2 and T6#5. (C) AAV2 capsid proteins analysis by Western blot. MW: molecular weight. Rep proteins were detected using an anti-Rep monoclonal antibody (Rep78, Rep68, Rep52, Rep40) (Acris Antibodies GmbH). Capsid proteins of AAV5 were detected using an anti-AAV-5 capsid (VP1, VP2, and VP3) polyclonal antibody from (Abnova). A monoclonal antibody recognizing the Cap2 proteins (VP1, VP2, VP3) supplied by antibody-enligne.fr was used.
3.3. Analysis of Sf-9 culture supernatants infected with recombinant baculovirus Sf-9 cells were grown in suspension in Sf-900 II medium and infected with the recombinant baculovirus (BacCap2, BacCap5, BacRep2, T2 BacRep2/Cap2 and T6 BacRep2/Cap6). Three days postinfection, the culture was stopped and the supernatants were harvested by centrifugation. To assess the composition of baculovirus particles supernatants were subjected to Western blotting using appropriate antibodies, Fig. 2 shows that the Cap proteins of AAV serotypes 2 and 5 were correctly expressed in Sf-9 cells. VP1, VP2 and VP3 have the correct molecular weight of 87 kDa, 73 kDa and 62 kDa, respectively. In addition, the Rep proteins (Rep78, Rep68, Rep52 and Rep40) were correctly expressed as shown by Western blot analysis (Fig. 2).
Fig. 3. Western blot analysis of HEK-293EBNA culture supernatant. MW: molecular weight marker, 24 h: culture supernatant collected after 24 h and concentrated 3.5-fold (−) cont: negative control corresponding to culture supernatant of cells transfected with rAAV5 containing GFP gene. A monoclonal antibody (Lifespan Biosciences) recognizing ORF2 region of the capsid protein of HEV (aa 434–457) was used as primary antibody.
3.4. BacHEVORF2 construction using the Baculogold system 3.6. Expression of HEVORF2 (112–660aa) protein The plasmid pMK-RQ containing the gene of HEVORF2 (aa 112–660 aa) was supplied by Geneart (Toronto, Canada). To construct the pVV1-HEVORF2, the plasmids pMK-RQ and pVV1GFP were digested with appropriate enzymes then purified from agarose gel. The ligation was performed under adequate conditions using the linearized pVV1 and HEVORF2 DNA. The presence of the insert was checked in recombinant colonies by PCR. The purified plasmid pVV1-HEV ORF2 112–660 and the linearized vector BacuGold were used for the co-transfection of Sf-9 cells. This technique allows recombination between the vector and the viral DNA that occurs within the cell, to produce the recombinant baculovirus BacITRHEVORF2. Virus titer was determined using various methods, the highest titer was obtained at passage 3 (P3) and was equal to 5.41 × 109 PFU/mL. 3.5. Production of rAAV5 containing HEVORF2 gene in Sf9 cells Sf-9 cells were grown in SF900-II medium to a cell density of 2 × 106 cells/mL, then infected with the three baculovirus (BacRep2, BacCap5 and BacITRHEVORF2) at an MOI of 1. Two days postinfection, cell viability was equal to 27.5%. rAAV was collected by centrifugation. Cell pellet was also collected to extract intracellular rAAV. The titer of rAAV was determined by q-PCR and was equal to 2.19 × 1011 vg/mL.
To check the expression of the protein HEVORF2, HEK293 EBNA cells were transduced with rAAV5 particles containing the transgene of interest and the adenovirus helper (Ad-5.VR1516) at an MOI of 60. Cells were incubated at 5% CO2 , 37 ◦ C for 24 h. Culture harvest was collected at 24 h post-infection and then analyzed by Western blot. The immunoreactive band observed at a molecular weight of 62 kDa (Fig. 3) corresponds to the protein of interest (HEVORF2 (112–660aa)). This result indicates that the protein HEVORF2 was correctly produced. Acknowledgements The authors gratefully thanks Dr Kotin laboratory (NIH, USA) for providing the baculovirus vectors, and The International Center for Genetic Engineering and Biotechnology (ICGEB, Trieste, Italy) for financial support. References [1] Tam AW, Smith MM, Guerra ME, Huang CC, Bradley DW, Fry KE, et al. Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 1991;185(1):120–31. [2] Emerson S, Purcell R, Hepatitis E. virus. In: Knipe DM, Howley PM, editors. Fields virology. fifth ed. Philadelphia: Lippincott Williams & Wilkins; 2007. p. 3047–58. [3] Aggarwal R. Hepatitis E: historical, contemporary and future perspectives. J Gastroenterol Hepatol 2011;(1):72–82.
Please cite this article in press as: Trabelsi K, et al. Development of a vectored vaccine against Hepatitis E virus. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.041
G Model JVAC-15126; No. of Pages 4 4
ARTICLE IN PRESS K. Trabelsi et al. / Vaccine xxx (2014) xxx–xxx
[4] Aggarwal R, Jameel S, Hepatitis E. Vaccine. Hepatol Int 2008;2:308–15. [5] Zhu FC, Zhang J, Zhang XF, Zhou C, Wang ZZ, Huang SJ, et al. Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase 3 trial. Lancet 2010;376: 895–902. [6] Graentzdoerffer A, Nemetz C. Titration of non-occluded baculovirus using a cell viability assay. BioTechniques 2003;34:260–4.
[7] Shen CF, Meghrous J, Kamen A. Quantitation of baculovirus particles by flow cytometry. J Virol Methods 2002;105:321–30. [8] Hopkins RF, Esposito D. A rapid method for titrating baculovirus stocks using the Sf-9 easy titer cell line. BioTechniques 2009;47:785–8. [9] Aucoin MG, Perrier M, Kamen A. Production of adeno-associated viral vectors in insect cells using triple infection: optimisation of baculovirus concentration ratios. Biotechnol Bioeng 2006;95:1081–92.
Please cite this article in press as: Trabelsi K, et al. Development of a vectored vaccine against Hepatitis E virus. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.041
ARTICLE IN PRESS
JVAC-15126; No. of Pages 4
Vaccine xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Development of a vectored vaccine against Hepatitis E virus Khaled Trabelsi a , Amine Kamen b , Héla Kallel a,∗ a Viral Vaccines Research & Development Unit, Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Institut Pasteur de Tunis, 13 Place Pasteur, BP 74 1002 Tunis, Tunisia b Animal Cell Technology, Biotechnology Research Institute, National Research Council of Canada, Montreal, Canada
a r t i c l e
i n f o
Article history: Available online xxx Keywords: Hepatitis E virus Insect cells SF9 Baculovirus Adeno-associated virus
a b s t r a c t Hepatitis E virus is a non-enveloped ssRNA virus [1] that causes human acute hepatitis through primarily fecal and oral transmission [2]. Currently, no commercial hepatitis E (HEV) vaccine is available. In the absence of an appropriate cell culture system for HEV propagation, HEV pseudocapsids (ORF2 protein) have been produced either in Escherichia coli or in insect cells and they have been shown to protect monkeys against virus challenge and to be effective in the prevention of natural HEV infection of humans. In this work, we propose to develop a novel candidate vaccine against hepatitis E infection using adenoassociated virus (AAV) as a vector expressing the gene of the truncated capsid protein of HEV (aa 112–aa 660). rAAV will be produced in Sf9 cells using the baculovirus expression vector system. For this purpose, construction of recombinant baculoviruses was performed and viral stocks of BacRep, BacCap for serotypes 2, 5 and 6 were prepared in Sf9 cells. The recombinant baculovirus coding for the truncated capsid protein of HEV (BacITRHEVORF2) was also constructed, the virus titer was equal to 5.41 × 109 PFU/mL, at the third passage. Transduction of HEK 293 EBNA cells with rAAV was carried out; the production of HEVORF2 was confirmed by Western blot. Optimization of rAAV production in Sf9 cells is currently ongoing. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction HEV is classified into the family Hepeviridae and genus Hepevirus. Based on the available nucleotide sequences, HEV isolates have been divided into five genotypes, each with a different geographical distribution. The HEV genome contains three open-reading frames (ORFs) that encode the ORF1 non-structural polyprotein with proposed biochemical functions, the ORF2 major viral capsid protein, and the ORF3 regulatory protein. Various recombinant forms of the ORF2, expressed using different heterologous systems are being explored [3]. Two candidate vaccines are in development and are being tested in clinical trials. The recombinant ORF2 proteins produced using baculovirus vectors in insect cells have been pursued vigorously as candidate vaccines. The 56-kDa ORF2 truncated protein expressed in insect cells, accumulates in the cytoplasm. This protein was found
∗ Corresponding author at: Viral Vaccines Research and Development Unit, Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Institut Pasteur de Tunis, 13 Place Pasteur, BP 74, 1002 Belvédère, Tunis, Tunisia. Tel.: +216 71 783 022; fax: +216 71 791 833. E-mail address: [email protected] (H. Kallel).
to be an efficient immunogen in monkeys when given with an alum adjuvant. Phases II–III efficacy trial conducted in young healthy adults (soldiers) in Nepal, showed an efficacy rate of 95%. The volunteers were given 20 g of the alum-adjuvanted recombinant HEV protein at 0, 1 and 6 months. However, despite such progress, the vaccine still needs several years more work before its licensing [4]. The second candidate vaccine is expressed in Escherichia coli, it is called HEV293, contains ORF2 amino acids 376–606. A large phase III trial of this vaccine was conducted in adults in China; the volunteers received three doses of HEV 239 (30 g of purified recombinant hepatitis E antigen adsorbed to 0.8 mg aluminum hydroxide suspended in 0.5 ml buffered saline) given intramuscularly at 0, 1, and 6 months. Data showed that this vaccine was safe and effective. Adverse effects attributable to the vaccine were few and mild. No vaccination-related serious adverse event was noted [5]. However both candidate vaccines need the use of an adjuvant, are delivered intramusculary and require several injections to be effective. Therefore these vaccines may have a high cost, and could not be affordable for populations living in developing countries, where HEV is endemic. In this work we suggest to develop a vectored vaccine against hepatitis E virus that will be delivered by the nasal route. For this purpose we will use the AAV as a vector to
http://dx.doi.org/10.1016/j.vaccine.2014.02.041 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Trabelsi K, et al. Development of a vectored vaccine against Hepatitis E virus. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.041
G Model JVAC-15126; No. of Pages 4
ARTICLE IN PRESS K. Trabelsi et al. / Vaccine xxx (2014) xxx–xxx
2
Fig. 1. Electrophoretic separation on 1% agarose gel. (A) T6 Rep2/Cap6 Bacmid clones #2 and #5. (B) T2 Rep2/Cap2 Bacmid clones #3 and #1. (C) BacCap5 Bacmid clones #8, #9, and #10. (Lad) DNA ladder.
deliver the antigen which is the truncated capsid protein of HEV (aa 112–aa 660). Baculovirus and insect cells platform will be used for the production of rAAV. 2. Material and methods
pH 7.4, 0.02% Tween-20, 3% skimmed milk). Membranes were incubated with the appropriate primary detection antibody for 1 h with gentle agitation, washed three times with PBST, then incubated for an additional 1 h with the appropriate peroxidase conjugated secondary antibody. Immunoreactive proteins were visualized by ECL (GE Healthcare, Uppsala, Sweden).
2.1. Cells and medium 3. Results and discussion Sf9 insect cells were used in this study. They were grown in suspension in the serum free medium SF900II (Invitrogen, The Netherlands) in polycarbonate Erlenmeyer flasks (Corning, NY, USA) at 27 ◦ C and 115–120 rpm. Cultures were seeded at 0.45–0.5 × 106 cells/mL. 2.2. Plasmid and recombinant baculovirus construction BacITRHEVORF2 was constructed using the Baculogold system from Becton Dickinson (USA). BacRep2, BacCap2, BacT2 Rep/Cap and BacT6 Rep/cap baculoviruses were constructed using the Bacto-Bac baculovirus expression system from Invitrogen (Canada). The plasmids pFBVP5, pFBDLSR Rep2, pSR657 BacT2 Rep/Cap, pSR646 Bac T6 Rep/Cap were kindly provided by Dr Kotin from the Laboratory of Biochemical Genetics (NHLB, NIH, USA). 2.3. Virus stock preparation Sf9 cells growing exponentially were infected at a density 2–2.5 × 106 cells/ml with 0.1–0.5% (v/v) of recombinant baculovirus. Virus harvests were collected after 72–96 h post-infection, culture supernatants were centrifuged at 4000 rpm, 10 min then stored at 4 ◦ C or −80 ◦ C in SF900II medium + 1% fetal bovine serum (FBS). 2.4. Estimation of virus titer Virus titer was estimated according to the MTT viability assay [6], flow cytometry [7] and the easy titer Sf9/GFP [8]. 2.5. AAV quantification Vector genome quantification was carried out by real timePCR [9]. 2.6. Western blot Samples were analyzed by SDS-PAGE under reducing conditions using 15% polyacrylamide gels and then transferred onto nitrocellulose membranes using the transfer System (Bio-Rad, USA). The membranes were then blocked overnight in PBST solution (PBS,
3.1. Construction of the vectors BacRep2, BacCap5, the dual plasmids T2 Rep2/Cap2 and T6 Rep2/Cap6 The plasmids provided by Dr Kotin were used to construct the different types of baculovirus (BacRep2, BacCap5, BacRep2/Cap2 and BacRep2/Cap6) using the Bac-to-Bac system. The expression cassette of the pFastBac vector recombines with the parent bacmid in DH10BacTM E. coli competent cells to form the expression bacmid. After extraction and purification the bacmid were analyzed by electrophoretic separation in agarose gels (Fig. 1). The bacmid were then transfected into insect cells for the production of recombinant baculovirus. 3.2. Production of recombinant baculovirus and titration Recombinant baculovirus (BacRep2, BacCap2, BacCap5, BacRep2/Cap2, BacRep2/Cap6 and BacITRHEVORF2) were amplified in Sf9 cells grown in SF900-II medium over three passages. Cells were grown at 27 ◦ C and infected at an MOI of 1 and at an initial cell density of 2 × 106 cells/mL. Once cell viability reached a level less than 60%, baculovirus were harvested by centrifugation. The harvests were also filtrated through 0.22 m, 1% fetal calf serum was added to the baculovirus stocks before being stored at −80 ◦ C. Virus titers were estimated using different methods; data obtained are shown in Table 1. Virus titer of the different baculovirus obtained at the third passage was slightly higher than 108 PFU/mL as determined by the MTT method.
Table 1 Titration of recombinant baculovirus using different methods. Baculovirus
TCID50 (PFU/mL) (using Sf-9 easy titer cell line)
FACS (PFU/mL)
MTT assay (PFU/mL)
P3 BacRep2/Cap2 P3 Bac Rep2/Cap6 P3 Bac Cap5 P3 BacCap2 P3 Bac Rep2
Nd Nd 7.64 × 107 Nd Nd
Nd Nd 1.97 × 109 Nd Nd
3.2 × 108 2.28 × 108 3.23 × 108 3.59 × 108 2.21 × 108
Nd, not determined.
Please cite this article in press as: Trabelsi K, et al. Development of a vectored vaccine against Hepatitis E virus. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.041
G Model JVAC-15126; No. of Pages 4
ARTICLE IN PRESS K. Trabelsi et al. / Vaccine xxx (2014) xxx–xxx
3
Fig. 2. (A) AAV5 capsid proteins analysis by Western blot. Detection of the Cap proteins (VP1, VP2, VP3) for clones #8, #9 and #10. (B) AAV2 replicase proteins (Rep78, Rep68, Rep52, Rep40) analysis by Western blot for clones #1, #8, T2#1, T6#2 and T6#5. (C) AAV2 capsid proteins analysis by Western blot. MW: molecular weight. Rep proteins were detected using an anti-Rep monoclonal antibody (Rep78, Rep68, Rep52, Rep40) (Acris Antibodies GmbH). Capsid proteins of AAV5 were detected using an anti-AAV-5 capsid (VP1, VP2, and VP3) polyclonal antibody from (Abnova). A monoclonal antibody recognizing the Cap2 proteins (VP1, VP2, VP3) supplied by antibody-enligne.fr was used.
3.3. Analysis of Sf-9 culture supernatants infected with recombinant baculovirus Sf-9 cells were grown in suspension in Sf-900 II medium and infected with the recombinant baculovirus (BacCap2, BacCap5, BacRep2, T2 BacRep2/Cap2 and T6 BacRep2/Cap6). Three days postinfection, the culture was stopped and the supernatants were harvested by centrifugation. To assess the composition of baculovirus particles supernatants were subjected to Western blotting using appropriate antibodies, Fig. 2 shows that the Cap proteins of AAV serotypes 2 and 5 were correctly expressed in Sf-9 cells. VP1, VP2 and VP3 have the correct molecular weight of 87 kDa, 73 kDa and 62 kDa, respectively. In addition, the Rep proteins (Rep78, Rep68, Rep52 and Rep40) were correctly expressed as shown by Western blot analysis (Fig. 2).
Fig. 3. Western blot analysis of HEK-293EBNA culture supernatant. MW: molecular weight marker, 24 h: culture supernatant collected after 24 h and concentrated 3.5-fold (−) cont: negative control corresponding to culture supernatant of cells transfected with rAAV5 containing GFP gene. A monoclonal antibody (Lifespan Biosciences) recognizing ORF2 region of the capsid protein of HEV (aa 434–457) was used as primary antibody.
3.4. BacHEVORF2 construction using the Baculogold system 3.6. Expression of HEVORF2 (112–660aa) protein The plasmid pMK-RQ containing the gene of HEVORF2 (aa 112–660 aa) was supplied by Geneart (Toronto, Canada). To construct the pVV1-HEVORF2, the plasmids pMK-RQ and pVV1GFP were digested with appropriate enzymes then purified from agarose gel. The ligation was performed under adequate conditions using the linearized pVV1 and HEVORF2 DNA. The presence of the insert was checked in recombinant colonies by PCR. The purified plasmid pVV1-HEV ORF2 112–660 and the linearized vector BacuGold were used for the co-transfection of Sf-9 cells. This technique allows recombination between the vector and the viral DNA that occurs within the cell, to produce the recombinant baculovirus BacITRHEVORF2. Virus titer was determined using various methods, the highest titer was obtained at passage 3 (P3) and was equal to 5.41 × 109 PFU/mL. 3.5. Production of rAAV5 containing HEVORF2 gene in Sf9 cells Sf-9 cells were grown in SF900-II medium to a cell density of 2 × 106 cells/mL, then infected with the three baculovirus (BacRep2, BacCap5 and BacITRHEVORF2) at an MOI of 1. Two days postinfection, cell viability was equal to 27.5%. rAAV was collected by centrifugation. Cell pellet was also collected to extract intracellular rAAV. The titer of rAAV was determined by q-PCR and was equal to 2.19 × 1011 vg/mL.
To check the expression of the protein HEVORF2, HEK293 EBNA cells were transduced with rAAV5 particles containing the transgene of interest and the adenovirus helper (Ad-5.VR1516) at an MOI of 60. Cells were incubated at 5% CO2 , 37 ◦ C for 24 h. Culture harvest was collected at 24 h post-infection and then analyzed by Western blot. The immunoreactive band observed at a molecular weight of 62 kDa (Fig. 3) corresponds to the protein of interest (HEVORF2 (112–660aa)). This result indicates that the protein HEVORF2 was correctly produced. Acknowledgements The authors gratefully thanks Dr Kotin laboratory (NIH, USA) for providing the baculovirus vectors, and The International Center for Genetic Engineering and Biotechnology (ICGEB, Trieste, Italy) for financial support. References [1] Tam AW, Smith MM, Guerra ME, Huang CC, Bradley DW, Fry KE, et al. Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 1991;185(1):120–31. [2] Emerson S, Purcell R, Hepatitis E. virus. In: Knipe DM, Howley PM, editors. Fields virology. fifth ed. Philadelphia: Lippincott Williams & Wilkins; 2007. p. 3047–58. [3] Aggarwal R. Hepatitis E: historical, contemporary and future perspectives. J Gastroenterol Hepatol 2011;(1):72–82.
Please cite this article in press as: Trabelsi K, et al. Development of a vectored vaccine against Hepatitis E virus. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.041
G Model JVAC-15126; No. of Pages 4 4
ARTICLE IN PRESS K. Trabelsi et al. / Vaccine xxx (2014) xxx–xxx
[4] Aggarwal R, Jameel S, Hepatitis E. Vaccine. Hepatol Int 2008;2:308–15. [5] Zhu FC, Zhang J, Zhang XF, Zhou C, Wang ZZ, Huang SJ, et al. Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase 3 trial. Lancet 2010;376: 895–902. [6] Graentzdoerffer A, Nemetz C. Titration of non-occluded baculovirus using a cell viability assay. BioTechniques 2003;34:260–4.
[7] Shen CF, Meghrous J, Kamen A. Quantitation of baculovirus particles by flow cytometry. J Virol Methods 2002;105:321–30. [8] Hopkins RF, Esposito D. A rapid method for titrating baculovirus stocks using the Sf-9 easy titer cell line. BioTechniques 2009;47:785–8. [9] Aucoin MG, Perrier M, Kamen A. Production of adeno-associated viral vectors in insect cells using triple infection: optimisation of baculovirus concentration ratios. Biotechnol Bioeng 2006;95:1081–92.
Please cite this article in press as: Trabelsi K, et al. Development of a vectored vaccine against Hepatitis E virus. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.041
